Multiple epitope fusion protein

ABSTRACT

Multiple epitope fusion proteins and immunoassays using the same are disclosed. The multiple epitope fusion proteins are encompassed by the general structural formula (A) x −(B) y −C 2  which represents a linear amino acid sequence, wherein B is an amino acid sequence of an epitope or cluster of epitopes and each B contains at least five and not more than 1,000 amino acids, y is an integer of 2 or more, A and C are each independently an amino acid sequence of an epitope or cluster of epitopes not adjacent to B in nature and x and z are each independently an integer of 0 or more wherein at least one of x and z is 1 or more.

This applications is a continuation of U.S. patent application Ser. No.08/653,226, filed May 24, 1996, now U.S. Pat. No. 6,514,731, from whichapplication priority is claimed pursuant to 35 U.S.C. § 120 and whichapplication is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the fields of protein synthesis andimmunoassays and specifically relates to methods of synthesizing longchains of amino acids that contain multiple copies of epitopes forviruses such as HCV, and to assay devices that utilize multiple epitopesto detect the presence of antibodies.

BACKGROUND OF THE INVENTION

In general, immunoassays are produced by first determining epitopes thatare specifically associated with a virus and then determining which ofthe epitopes is preferred for the assay being developed. When theimmunodominant epitopes are isolated, their sequences are determined,and genetic material for producing the immunodominant epitopes isproduced. Methods of producing proteins by either chemical or biologicalmeans are known, as are assays used to detect the presence of antibodiesto particular epitopes.

In producing immunoassays the overall object is to obtain an immunoassaywhich is both highly sensitive and highly selective. More specifically,the immunoassay must be designed such that it can detect even very lowlevels of the material it is designed to detect, i.e., it is highlysensitive. An assay having a high degree of sensitivity ensures that asample, which has been tested, is not contaminated with the material theassay is designed to detect. For example, a highly sensitive assay thatdetects even the slightest presence of antibodies for a given virus isdesirable in that it makes it possible to detect and thus discardsamples that contain any amount of the antibody indicating that thesamples contain the virus.

Although a high degree of sensitivity is desirable in an assay, it isnot desirable if the assay is falsely indicating the presence of thematerial, i.e. the assay is providing a false positive result. Suchfalse positive results can occur when the analyte has a high degree ofsimilarity with another material present in the sample. The ability onan assay to differentiate between two similar but different materialsrelates to its selectivity.

An immunoassay with a high degree of selectivity will detect thepresence of a material being assayed for even when that material ispresent in the sample in combination with other materials having asimilar structure. Thus, a highly selective immunoassay will eliminatemost false positive results. In general, as selectivity increasessensitivity decreases. This occurs, in part, due to the high degree ofvariability in viruses. Assays which are designed to be highly sensitivemust take into account the high degree of variability between differentviruses. As virus variability is accommodated to improve sensitivity,the selectivity decreases. Alternatively, as one produces an immunoassaythat is more and more selective with respect to a particular virus, thelikelihood of the assay becoming so selective as to have decreasedsensitivity increases.

To a large extent the problem of providing for improved selectivity(less false positives) is dealt with by searching for and finding themost immunodominant epitopes. The problem of sensitivity (lowconcentration detection) is dealt with by providing immunodominantepitopes from a variety of different regions of the virus.

Current assays are designed to utilize relatively few peptides selectedas “major epitopes” or highly immunodominant epitopes. The assaysensitivity is dependent on the number of major epitopes available onthe solid support. If the availability of epitopes is limited by thenumber of peptides that can be coated on the solid phase, then thatassay will have reduced sensitivity. These results can be demonstratedas poor assay dilution sensitivity and poor seroconversion sensitivitiesand/or false negative determinations (Chien, D. Y. et al. (1993) J.Gastroenterology and Hepatology 8:S33–39).

There is currently a need to improve the sensitivity and selectivity ofassays for antibodies to pathogens in biological fluids and therebyimprove diagnosis of pathogen infection resulting in improved screeningof blood supplies.

SUMMARY OF THE INVENTION

Multiple copy fusion antigen (MEFA) immunoassays capable of detectingantibodies from multiple strains of a pathogen in a single assay areproduced by (1) identifying nucleotide sequences that encode a pluralityof different epitopes, including immunodominant components; (2) placingthe nucleotide sequences into an expression cassette wherein at leasttwo copies of a sequence coding for the same epitope region of anorganism such as virus or corresponding regions of different strains ofthe virus is placed in a single cassette; (3) transforming a suitablehost with one or more copies of the cassette in order to expresssequences encoding epitopes, which sequences will include two or morecopies of at least one epitope in a single chain antigen; (4) purifyingthe expressed multiple epitope antigen; and (5) adapting the purifiedmultiple epitope antigen for an immunoassay, where adapting may include,but is not limited to, the following: coating the multiple epitopeantigen on a surface of a substrate; covalently attaching a detectablemarker to the multiple epitope antigen; and the like. The purifiedepitopes are encompassed by the general structural formula(A)_(x)−(B)_(y)−(C)_(z) which represents a linear amino acid sequence. Bis an amino acid sequence of at least five and not more than 1,000 aminoacids of an antigenic determinant or cluster of antigenic determinants,and y is an integer of 2 or more. Each copy of B is an equivalentantigenic determinant (for example, each copy is an epitope from adifferent viral strain). A and C are each independently an amino acidsequence of an epitope or cluster of epitopes not immediately adjacentto B in nature; and, x and z are each independently an integer of 0 ormore, wherein at least one of x and z is 1 or more. Preferably the yepitopes of B are equivalent antigenic determinants from different viralstrains thereby increasing the variety of pathogens detectable by asingle multiple epitope antigen. The selectivity is further improved byincluding immunodominant epitopes from the same region of two or moredifferent strains of the same virus. More preferably, the equivalentantigenic determinants of B have different serotype specificity.Homology between the B epitopes is at least 30%, preferably at least40%. The epitopes of the invention are more soluble, and are thereforemore easily purified, than conventional epitopes. Further, the presenceof repeating epitope sequences (1) decreases masking problems and (2)improves sensitivity in detecting antibodies by allowing a greaternumber of epitopes on a unit area of substrate. Sensitivity is furtherimproved by placing the multiple copy epitopes of the invention on smallspherical or irregularly shaped beads or microparticles therebyincreasing the exposed surface area per given area of an assay device.

An object of the invention is to provide an amino acid sequencecomprised of a plurality of epitopes wherein at least the antigenicdeterminant portion of at least one of the epitopes is repeated two ormore times.

Another object of the invention is to provide a method of producing animmunoassay using multiple epitope fusion antigens.

A feature of the invention is that amino acid sequences that comprisemultiple copies of a given epitope sequence have higher solubility ascompared with amino acid sequences comprising only a single copy of anygiven epitope.

Another feature of the invention is that the nucleotide sequencesencoding the epitopes are in a linear order that may be different fromtheir linear order in the genome of the pathogen. Thus, the antigenicdeterminants of A, B, and C may be in a linear order different from thenaturally occurring antigenic determinants of A, B and C. The linearorder of the sequences of the invention is preferably arranged foroptimum antigenicity of the expressed amino acid sequences comprisingthe multiple epitope fusion antigen.

An advantage of the invention is that the multi-epitope antigens offormula (I) can be more easily purified as compared with conventionalepitopes.

Another advantage of the invention is that masking of an antigenicdeterminant can be reduced.

Another advantage of the invention is that the immunoassays utilizingthe multiple epitope fusion antigens have improved sensitivity andselectivity.

Another advantage of the invention is that the multiple epitopes,particularly the repeated epitopes of B, provide an assay capable ofdetecting more than one pathogen or more than one strain of a singlepathogen based on the type specificity of the epitopes.

Another feature of the invention is that the multiple epitope sequencesof formula (I) can be designed to include a larger number and or longersequences than are generally present on epitope sequences containingonly a single copy of any given epitope.

Another advantage of the invention is that the design of themulti-epitope antigens as per formula (I) makes it possible to include agreater number of antigenic determinants on a unit area of surface of animmunoassay as compared to antigens containing only a single copy of anygiven epitope.

The invention also provides the advantage of improving the generalspecificity and sensitivity of serological tests when multiple epitopesare required and solid phase surface area is limiting. Additionally,immunoassay tests based on a single chimeric antigen will greatlysimplify the manufacturing process, particularly for tests which requireantigens labelled with detectable markers.

An embodiment of the invention further provides a rapid capture ligandimmunoassay using multiple epitope fusion antigens that is simple andconvenient to perform because it is a one step simultaneous assay.Detection is by the attachment of a detectable marker to a member of theantigen/antibody complex, preferably to the antigen. Attachment may beby covalent means or by subsequent binding of detectably labeledantibodies, such as a standard sandwich assay, or by enzyme reaction,the product of which reaction is detectable. The detectable marker mayinclude, but is not limited to, a chromophore, an antibody, an antigen,an enzyme, an enzyme reactive compound whose cleavage product isdetectable, rhodamine or rhodamine derivative, biotin, strepavidin, afluorescent compound, a chemiluminescent compound, such as dimethylacridinium ester (DMAE, Ciba Corning Diagnostics Corp.), derivativesand/or combinations of these markers.

In another embodiment of the invention, the capture ligand format assaycontains a MEFA as an antigen, as well as, an additional detectableepitope added to the assay mixture. The additional detectable epitopemay be a single epitope or multiple epitopes and may include, but is notlimited to, the epitopes included in the MEFA, preferably epitopes fromregions such as E1, E2 and c33c. According to this embodiment of theinvention, the additional epitope is attached or attachable to adetectable marker as described above. Where the additional epitope haspreferred characteristics such as conformation, glycosylation, and thelike, the additional epitope is expressed as a recombinant polypeptidefrom a cell, which expression provides the epitope in a desired form.Preferably the epitope is obtainable from the cell using gentleisolation conditions that preserves the desired characteristics of theepitope. The cell may be any appropriate cell such as a mammalian cell,preferably a chinese hamster ovary (CHO), or a bacterial, yeast orinsect cell from which the additional epitope can be isolated in thedesired form.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the multiple copy epitopes, immunoassays, andmethods for producing such as more fully set forth below, with referencebeing made to the accompanying general structural formula forming a parthereof wherein like symbols refer to like molecular moieties throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing the identification, amino acidsencoded, and the arrangements of epitopes in MEFA-3, MEFA-5, and MEFA-6.

FIG. 2 is a schematic drawing showing the MEFA-5 antigen epitopes andtheir location within the HCV genome. A diagram of the expression vectorfor MEFA-5 is also provided.

FIG. 3 is a schematic drawing showing the MEFA-6 antigen epitopes andtheir location within the HCV genome. A diagram of the expression vectorfor MEFA-6 is also provided.

FIG. 4 is a schematic drawing of an enzyme-linked immunosorption assay(ELISA) in which a MEFA is adsorbed onto the surface of a solid support.

FIG. 5 is a schematic diagram of an antibody capture format fordetection of anti-HCV antibodies by chemiluminescence in which a MEFA isattached to a detectable marker molecule, DMAE. Also indicated is aformat in which a MEFA (MEFA-6) and an additional epitope (c33c) are theantigens of the assay.

FIG. 6 is a schematic diagram of an antibody capture format fordetection by chemiluminescence of human anti-pathogen antibodies inwhich an antigen (MEFA) is attached to biotin (B) that binds strepavidinlabeled with DMAE.

FIG. 7 is a plot comparing the dilution sensitivity of MEFA-6-DMAE andMEFA-6-DMAE +c33c-DMAE to the dilution sensitivity of a commercialELISA, HCV 2.0G (second generation) ELISA.

FIG. 8 is a plot comparing the seroconversion sensitivity of acommercial ELISA (Abbott Laboratories), MEFA-6, MEFA-6+c33c, and RIBA®3.0. Samples were taken from a chronically infected patient over time(bleed dates).

FIG. 9 is a diagram correlating HCV antibody detection (positive ornegative) in samples by HCV Second Generation ELISA to detection by MEFACLIA.

FIG. 10 is a chart illustrating the accuracy of the MEFA-6-DMAEchemiluminescence immunoassay (CLIA) of the invention. All knownnegative samples exhibited relative light units (RLU) below the cutoffvalue, while known positive samples exhibited RLUs well above the cutoffvalue.

DETAILED DESCRIPTION OF EMBODIMENTS

Before the present multiple epitope fusion proteins, immunoassays andmethods for producing and using such are described, it is to beunderstood that this invention is not limited to the particular aminoacid sequences, immunoassays or methods of production as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting since the scope of the present inventionwill be limited only by the appended claims.

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of disclosing anddescribing the particular technology which the publication is cited inconnection with.

Definitions

The term “multiple copy” shall mean that a sequence of amino acids whichcontains at least five and not more than 1,000 amino acids in a linearfashion is repeated two or more times within a linear molecule. Therepeating sequence need not be directly connected to itself, is notrepeated in nature in the same manner, and further may be present withina larger sequence which includes other amino acids not repeated or“copied.” The sequence of at least five and not more than 1,000 aminoacids comprises an epitope as defined below. For the purposes of thisinvention, a “copy” of an amino acid sequence may be either an exactsequence copy or a sequence which corresponds to the same epitope of adifferent viral strain, i.e. copies are either exact copies or sequenceswhich are “equivalent antigenic determinants” as defined below.

The term “epitope” shall mean a sequence of at least five, and not morethan 1,000 acids connected in a linear fashion, which amino acids, bythemselves or as part of a larger sequence, bind to an antibodygenerated in response to such sequence.

The term “conformational epitope” shall mean a recombinant epitopehaving structural features native to the amino acid sequence encodingthe epitope within the full length natural protein. Native structuralfeatures include, but are not limited to, glycosylation and threedimensional structure. Generally, a conformational epitope is added tothe MEFA-containing immunoassay mixture to enhance assay sensitivity andselectivity. Preferably, a recombinant conformational epitope isexpressed in a cell from which it is extractable under conditions whichpreserve its desired structural features, e.g. without denaturation ofthe epitope. Such cells include bacteria, yeast, insect, and mammaliancells. Preferably, the cell in which a conformational epitope isexpressed is a mammalian cell, such as a chinese hamster ovary cell(CHO). Expression and isolation of recombinant conformational epitopesfrom the E1 and E2 regions of HCV are described in WO 96/04301, WO94/01778, WO 95/33053, WO 92/08734, which applications are hereinincorporated by reference in their entirety.

The term “expression cassette” shall mean a DNA sequence which containsa coding region operably linked to suitable control sequences capable ofeffecting expression of the coding region in a compatible host.Expression systems invariably comprise a promoter, but, depending on thehost intended, may contain additional critical DNA such as ribosomebinding site or CAP site, termination sequence, and optional enhancersequences upstream from the promoter or in other operable locations. Therecombinant expression cassettes of the invention herein comprise a DNAof the invention encoding a MEFA operably linked to additional DNAsequences that are capable of effecting its expression. The expressioncassette may reside on a transfer vector such as a plasmid or othervector that is self-replicating independently of the chromosome of thehost cell, or may be constructed so that when inserted into a host cellit is able to integrate into the chromosome.

The term “equivalent antigenic determinant” shall mean an antigenicdeterminant from different sub-species or strain of a given organisme.g., a different strain of a virus such as strains 1, 2, and 3 ofhepatitis C virus. More specifically for a virus such as hepatitis C,epitopes are known, such as 5-1-1, and such epitopes vary between theknown strains 1, 2, and 3. Thus, the epitope 5-1-1 from the threedifferent strains are equivalent antigenic determinants and thus are“copies” even though their sequences are not identical. In general theamino acid sequences of equivalent antigenic determinants will have ahigh degree of sequence homology, e.g., amino acid sequence homology ofmore than 30%, preferably more than 40%.

The term “tracer” shall mean any detectable marker molecule attachableto an epitope or a MEFA. Attachment is preferably by covalent means.Detectable marker molecules useful as tracers in the invention include,but are not limited to, dimethyl acridinium ester (DMAE), a chromophore,biotin, strepavidin, an antibody, an antigen, enzymes fluorogeniccompounds, rhodamine compounds, fluorescein, FITC, and the like.

Producing Immunoassays-General

Highly sensitive and selective immunoassays can be produced using themultiple epitope fusion antigens of the present invention. In order toproduce such immunoassays it is first necessary to identify a target forwhich a sample is to be assayed, e.g., assay for a particular virus in abody fluid sample. After identifying the virus of interest, thepreferred immunodominant epitopes of the virus are isolated, sequencedand nucleotide sequences encoding the amino acid sequences of theepitopes are determined and produced. The nucleotide sequences encodingthe amino acid sequences can be fused together using standardrecombinant methodology.

The fused sequence must include at least two copies of nucleotidesequences that encode a given epitope. The nucleotide sequence is thenplaced within an expression cassette and a suitable host is transformedwith the cassette. The host is allowed to express the sequences toprovide the multiple copy epitopes (multiple epitope fusion antigen,MEFA). The multiple copy epitopes produced are then purified, forexample, by affinity chromatography, which process is expedited to acertain degree due to the presence of the multiple copies of a givenepitope. The purified MEFAs are then coated onto the surface of thesubstrate for ELISA-type assays. Alternatively, the purified MEFAs areattached to a detectable marker tracer molecule for detection ofantibody binding, such as in a chemiluminescence assay (CLIA).

The essence of the invention is the purified multiple copy epitopes,i.e., purified fusion proteins that include multiple copies of a givenepitope fused, in a linear fashion in nature, to other epitopes that arenot normally connected to each other in this fashion (MEFAs). Thepurified epitopes are encompassed by the general structural formula (I)as follows: (A)_(x)−(B)_(y)−(C)_(z), which represents a linear aminoacid sequence, B is an amino acid sequence of an epitope or cluster ofepitopes and each B contains at least five and not more than 1,000 aminoacids, y is an integer of 2 or more, A and C are each independently anamino acid sequence of an epitope or cluster of epitopes not immediatelyadjacent to B in nature, and x and z are each independently an integerof 0 or more wherein at least one of x and z is 1 or more. When each ofx, y, or z is greater than 1 or when each of x, y, and z are greaterthan 1, the multiple copies of A, B and C may be identical, i.e., eachcopy of A (different from B and C) is the exact same amino acidsequence, each copy of B (different from A and C) is the exact sameamino acid sequence, and each copy of C (different from A and B) is theexact same amino acid sequence. Alternatively, each A, B or C copy maybe an equivalent antigenic determinant from different strains of thesame virus. Thus, for example, if y is 3, each B may be an identicalamino acid sequence or three different sequences from equivalentantigenic determinants from HCV strain 1, 2, and 3. The invention mayutilize genetic material encoding known epitopes or groups of epitopesby connecting the material in a nucleic acid construct that produces amultiple copy epitope of the formula (I).

HCV antibody capture assays in which the individual single epitopes arecoated on a solid support are less sensitive than capture assays inwhich a chimeric multiple epitope polyprotein, such as (C25) containingepitopes from the immunodominant core, c33c (NS3), and c1000 (NS4)region sequences (Chien, D. Y., et al (1992) Proc. Natl. Acad. Sci. USA89:10011–10015, herein incorporated by reference), is coated on a solidsupport. In turn, a capture assay using the C25 chimeric polyprotein isless sensitive than an HCV antibody capture assay using a MEFA of theinvention, which MEFA contains multiple copies of at least one epitopeand at least one copy is from a different HCV strain. Thus, a preferredMEFA of the invention having the general formula Ax-By-Cz, contains morethan one copy of an epitope (i.e., y is an integer of 2 or more), and atleast one of the epitopes of B is a different equivalent antigenicdeterminant (e.g. an epitope from a different pathogen strain).

The invention disclosed herein utilizes recombinant DNA technology andprotein engineering to design a recombinant polyprotein which fuses avariety of different immunodominant epitopes from a variety of pathogensor pathogen strains as the chimeric antigen for immunoassay development.Further, the invention utilizes multiple copies of selected epitopesfrom structural as well as non-structural coding regions of a genecombined and expressed as a recombinant polyprotein to significantlyimprove the sensitivity and selectivity of an immunoassay.

Epitopes used in making a multiple copy epitope of the invention can befrom a variety of different organisms. For example, the epitope may bean amino acid sequence from bacteria, protozoa, virus, rickettsiae,parasite or fungus. A preferred embodiment of the invention usesepitopes that are extracted from a bacteria or virus, with particularlypreferred epitopes being those obtained from a virus, such as from humanimmunodeficiency virus and, most preferably, from hepatitis C virus.

It is well known that any given organism varies from one individualorganism to another and further that a given organism such as a viruscan have a number of different strains. For example, hepatitis C virusincludes at least strains 1, 2, and 3. Each of these strains willinclude equivalent antigenic determinants. More specifically, eachstrain will include a number of antigenic determinants that will bepresent on all strains of the virus but, will be slightly different fromone viral strain to another. For example, hepatitis C includes theantigenic determinant known as 5-1-1 (in the NS3 region of the viralgenome). This particular antigenic determinant appears in threedifferent forms on the three different viral strains of hepatitis C.Accordingly, in a preferred embodiment of the invention all three formsof 5-1-1 appear on the multiple epitope fusion antigen of the invention.A MEFA of the invention has the above structural formula I, wherein y is3 and thus each of the three “Bs” are equivalent antigenic determinantsof 5-1-1 taken from the three different viral strains of hepatitis C.

The multiple copy epitope of the present invention can also includemultiple copies which are exact copies of the same epitope. For example,it is desirable to include two copies of an epitope from the core regionof hepatitis C. A particularly preferred embodiment of the presentinvention is the multiple copy epitope as shown within FIG. 3. Thismultiple copy epitope includes two exact copies of an epitope from thecore region and three copies of an epitope from the 5-1-1 region, whichcopies are equivalent antigenic determinants meaning that they areantigenic determinants taken from the three different viral strains ofhepatitis C. In general, equivalent antigenic determinants have a highdegree of homology in terms of amino acid sequence which degree ofhomology is generally 30% or more or more preferably 40% or more.

Producing HCV Immunoassays

Highly selective and sensitive immunoassays generally contain majorimmunodominant epitopes of the pathogen suspected of infecting apatient. Previously, immunoassays made use of individual epitopes tobind anti-HCV antibodies in biological samples.

For the virus HCV, major immunodominant linear epitopes were identifiedfrom the core, NS3 (nonstructural), NS4, and NS5 regions of the viruspolyprotein. Sallberg et al. assayed HCV core protein and putativematrix proteins against human serum samples containing antibodies to HCVand defined several immunodominant regions within the HCV proteins(Sallberg, M. et al. (1992) J. Clin. Microbiol. 30:1989–1994). Proteindomains of HCV-1 polyproteins including domains C, E1, E2/NS1, NS2, NS3,NS4, and NS5 were identified and their approximate boundaries providedby Chien and Rutter (Chien, D. Y. and Rutter, W., WO 93/00365,international publication date Jan. 7, 1993, herein incorporated byreference in its entirety). Kotwal et al. designed individualpolypeptides having sequences derived from the structural region of HCVin order to obtain an immunodominant epitope useful in testing sera ofHCV patients (Kotwal, G. J., et al. (1992) Proc. Natl. Acad. Sci.89:4486–4489).

Serologically definable subtypes of HCV were identified by Chien et al.as viral subtypes exhibiting varied antigenicity (presented at the ThirdInternational Hepatitis Meeting, Tokyo, May, 1993 and in Chien, D. Y. etal. (1994) Viral Hepatitis and Liver Disease, pp. 320–324, hereinincorporated by reference in its entirety). HCV-1 core, NS4, and NS5regions were found to contain serotype-specific epitopes. Individualputative core proteins from HCV-1 and HCV-2 were used as individualantigens to produce antibodies for enzyme-linked immunosorbent assays todetect HCV infection using serologically distinguishable core antigensubtypes (Machida, A. et al. (1992) Hepatology 16:886–891). Simmonds etal. investigated the effect of sequence variability between differenttypes of HCV upon the antigenicity of the NS4 protein by epitope mappingand by enzyme-linked immunosorbent assay (ELISA). These authors mappedtwo major antigenic regions in the HCV NS4 polyprotein that wererecognized by antibody elicited upon natural infection by HCV.Type-specific antibody to particular HCV types was also detected(Simmonds, P. et al. (1993) J. Clin. Microbiol. 31:1493–1503). Ching etal. prepared a series of synthetic peptides based on the sequence of ahighly conserved region of the HCV putative nucleocapsid (core) proteinand found an immunodominant region that was recognized by human andchimpanzee sera (Ching, W.-M. et al. (1992) Proc. Natl. Acad. Sci.89:3190–3194).

Assays involving single epitopes as test antigens have the disadvantagethat it is difficult to control solid phase coating of the supportsurface by large numbers of individual epitopes containing shortpeptides. In such cases where the assay involves deposition of animmunogenic antigen on a solid support, the sensitivity of the assay islimited by the amount of antigen that can be coated on the surface ofthe solid support.

An example of an immunoassay that includes immunodominant epitopes fromdifferent regions of a single virus subtype is disclosed within Chien etal. (Proc. Natl. Acad. Sci. USA 89:10011–10015 (1992), hereinincorporated by reference). The assay described by Chien utilizesrecombinant HCV polypeptides derived from many different regions of theHCV type 1 polyprotein, including that of chimeric recombinantpolyprotein, C25, comprises immunodominant components evident in boththe structural and non-structural regions. The polyproteins produced arerecombinantly derived viral polypeptides and are included on the surfaceof an immunoassay in order to capture antibodies, i.e., detect thepresence of antibodies generated in response to infection with HCV.However, these polyproteins contain epitopes from a single viral strainthereby limiting the ability to detect anti-HCV antibodies fromdifferent strains of the virus.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the multiple copy epitopes and immunoassays of the invention aswell as use such and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g. amounts, temperature, etc.)but some experimental error and deviation may be inherent in thedescription. Unless indicated otherwise, parts or parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade and pressure is at or near atmospheric.

Example 1 Construction and Expression of an HCV Epitope PolyproteinExpression Cassette

The following example illustrates the concept of preparing a polyproteincassette of major epitopes, particularly a cassette of multipleepitopes. The example further illustrates the success of using epitopesfrom different strains of a pathogen. It is also shown that ahydrophilic multiple epitope antigen increases the solubility of thepolyprotein. The epitopes are shown to maintain their native localconformation for binding to antibodies as evidenced by the antigenicityof the polyprotein.

The polyprotein expressed from the multiple epitope cassette is referredto herein as a Multiple Epitope Fusion Antigen (MEFA).

Preferably, where an epitope is repeated, the extra copy or copies aretandemly arrayed in the same orientation. It is understood that theregion of a viral coding sequence used as an epitope may be variedslightly and still retain antigenic activity, and that the amino acidnumbering designation may vary from strain to strain. Thus, the repeatedepitopes may vary one from another in amino acid sequence due to strainsequence variations and/or numbering designation. Preferably, the aminoacid sequences of repeated epitopes within a MEFA are at least 30%homologous at the amino acid level, more preferably at least 40%homologous at the amino acid level.

Unique restriction enzyme sites were introduced in order to connect theepitopes in the prescribed order and enhance the usefulness of theinvention by facilitating modifications in design of a chimeric antigen.The choice of restriction enzyme sites and cloning procedures arereadily determined by one of ordinary skill in the art of recombinantDNA technology. Preferably, the epitope junctions (amino acid sequencescreated between epitopes due to cloning) do not generate non-specificepitopes. Non-specific epitopes are, for example, non-HCV sequenceswhich do not exist adjacent to the HCV epitopes in nature. Non-specificepitopes may bind antibodies in a test sample causing false positiveassay results. Preferably, the multiple epitope fusion protein is testedfor false positive results due to such sequences generated at theepitope junctions. To avoid non-specific interactions with the MEFA dueto junction sequences, the DNA sequence encoding the junction may, forexample, be mutated such that (1) non-specific interactions with themutant amino acid sequence are reduced, and (2) cloning of the epitopefragments is possible.

Construction of a MEFA Expression Cassette of HCV Epitopes

The HCV MEFA-3 expression cassette was constructed by cloning the codingnucleotide sequences containing major epitopes in a tandem array asshown in FIG. 1. A major epitope was chosen based on antibody reactionfrequency and reaction intensity (titer) to the epitope (Chein, D. Y. etal. (1994) Viral Hepatitis and Liver Disease, pp. 320–324). The variousDNA segments coding for the HCV epitopes were constructed by PCRamplification or by synthetic oligonucleotides. The amino acid codonsencoded in each segment are shown below each segment. The complete HCV-1amino acid sequence (3011 amino acids) was determined by Choo, et al.(1991) PNAS, USA, 88:2451–2455, herein incorporated by reference in itsentirety. Oligonucleotides capable of binding to HCV are described inU.S. Pat. No. 5,350,671, herein incorporated by reference in itsentirety. The numbering of the amino acids in epitopes of the inventionfollows the numbering designation provided in Choo, et al., supra, inwhich amino acid #1 is the first methionine encoded by the codingsequence of core region. For example, an epitope segment from the coreregion is encoded by amino acid codons 10 to 53 of the HCV core protein.An epitope from the c33c region is encoded by amino acid codons 1192 to1457. The MEFA-3 construct contains in the expression cassette twocopies of the core segment epitope amino acids 10–35; one copy of thec33c epitope segment from amino acids 1192–1457; three copies ofequivalent antigenic determinants from the HCV NS4 region, specificallythe 5-1-1 region, where two of the epitopes are a segment (amino acids1694 to 1735) from the NS4 5-1-1 region of HCV type 1, while one copy issegment from the NS4 5-1-1 region of HCV type-2 (Nomoto) from aminoacids 1694 to 1735; two copies of the NS4 (C100) C-terminal region majorepitopes from amino acids 1901–1940; and two copies of major epitopesfrom amino acids 2278–2310 of the NS5 region. The MEFA-3 expressioncassette has the general structural formula 2-1-2-1-2-2 for Ax-By-Cz,where A=core-core-c33c, x=1; B=(5-1-1), y=3; andC=(c100)-(c100)-(NS5)-(NS5), z=1.

Other HCV MEFAs include MEFA-5 and MEFA-6 for which expression cassetteswere constructed by cloning the coding nucleotide sequences containingmajor epitopes in a tandem array as shown in FIG. 2 and Table 1, andFIG. 3 and Table 2, respectively. It is noted that all of the epitopesin MEFA-5 and MEFA-6 are from HCV type 1 except for two of the threeequivalent antigenic determinants of the 5-1-1 epitope. Epitopes fromthe 5-1-1 region have been found to vary between serotypes of HCV. Acopy of each of the HCV type-specific 5-1-1 epitopes present in theMEFAs described herein allows binding of any of the HCV types that maybe present in the test biological sample. It is a feature of theinvention that an epitope useful in distinguishing serotypes of a virussuch as HCV is provided as repeated equivalent antigenic determinants inorder to detect multiple types of a virus or pathogen in a single assay.Methods of determining HCV serotype are found in WO 96/27153, hereinincorporated by reference in its entirety.

TABLE 1 MEFA-5 Antigen Epitopes and Their Location Within the HCV Genomemefa aa# 5′ End Site Epitope HCV aa# Strain  1–154 NcoI hSOD 159–202EcoRI core  10–53 1 205–246 SacI core  10–53 1 251–268 PstI E1  303–3201 271–309 HindIII E2  405–444 1 310–576 DraIII c33c 1192–1457 1 579–625SphI 5-1-1 1689–1735 1 628–674 NruI 5-1-1 1689–1735 3 677–723 ClaI 5-1-11689–1735 2 726–765 AvaI c100 1901–1940 1 768–803 XbaI NS5 2278–2313 1806–841 Bg/II NS5 2278–2313 1

TABLE 2 MEFA-6 Antigen Epitopes and Their Location Within the HCV Genomemefa aa# 5′ End Site Epitope HCV aa# Strain  1–154 NcoI hSOD 159–176EcoRI E1  303–320 1 179–217 HindIII E2  405–444 1 218–484 DraIII c33c1192–1457 1 487–533 SphI 5-1-1 1689–1735 1 536–582 NruI 5-1-1 1689–17353 585–631 ClaI 5-1-1 1689–1735 2 634–673 AvaI c100 1901–1940 1 676–711XbaI NS5 2278–2313 1 714–749 Bg/II NS5 2278–2313 1 750–793 NcoI core 10–53 1 796–839 SacI core  10–53 1

The cloning procedures for preparation of MEFA-5 and MEFA-6 were similarto those used for preparing MEFA-3, above. MEFA-5 and MEFA-6 containedepitopes from the core, envelope, NS3, NS4 and NS5 regions of thehepatitis C polyprotein, including equivalent antigenic determinantsfrom HCV strains 1, 2, and 3. The various DNA segments coding for theHCV epitopes were constructed by PCR amplification or by syntheticoligonucleotides. FIGS. 2 and 3 are diagrammatic representations showingthe location of epitopes within the HCV genome used in MEFA-5 and -6, aswell as the MEFA vector construction. Tables 1 and 2 describe the aminoacid segments of each epitope, the linear arrangement of the variousepitopes and the number of copies in the MEFA-5 and MEFA-6 cassettes,respectively. The amino acids between each epitope (junction aminoacids) are derived from the restriction sites used for cloning.Preferably, a MEFA is tested in an immunoassay for a false positiveresult due to the non-pathogen (HCV, for example) junction amino acids.MEFA-5 differs from MEFA-6 in linear arrangement in that the coresegments are near the N-terminus in MEFA-5, but are at the C-terminus inMEFA-6. As the amino acid 10–53 core epitope is highly antigenic, itsplacement at the C-terminus improves the antigenicity of the MEFApossibly by improved interaction of epitopes from core proteins andantigens from me E1 and E2 regions with the anti-HCV antibodies of thesample.

The epitopes were subcloned into a yeast expression vector and thesequences verified before assembling the entire fusion antigen as anEcoRI-SalI fragment of 2060 bp (for MEFA-5 and MEFA-6) and as anEcoRI-SalI fragment of approximately 1927 bp (for MEFA-3). MEFA-5 andMEFA-6 were cloned as SOD fusion proteins (157 amino acids of humansuperoxide dismutase (hSOD)) under the regulation of the hybridADH₂-GAPDH promoter. The MEFA-5 and MEFA-6 expression cassettes wereligated to the yeast shuttle vector pAB24 (Chiron Corporation) toproduce pMEFA-5 and MEFA-6, as shown in FIG. 2 and FIG. 3, respectively.

As shown in FIG. 3, the MEFA-6 antigen includes multiple copies of HCVepitopes from the core and NS5 region; different serotype epitopes fromthe NS4 5-1-1 region; a single copy of major linear epitopes from thec100 C-terminal regions, E1, and E2 regions, as well as the HCV NS3(c33c) region. The general structural formula for MEFA-6 ishSOD-E1-E2-c33c-5-1-l(type 1)-5-1-1(type 3)-5-1-1(type 2)-c100-NS5(2copies)-core(2 copies). This antigen has a very high expression level inyeast, purifies to a high degree of homogeneity, and exhibits highsensitivity and high selectivity in the immunoassays described below.

Expression of a MEFA in Yeast

The following example of the expression of a MEFA in yeast may beapplied to the expression of any MEFA of the invention. It is within thescope of the instant invention to vary the conditions, as necessary, toexpress a particular MEFA construct. Cells preferred for expression of aMEFA of the invention include bacteria, yeast, and insect cells. Theexpression of MEFA-6 in yeast is provided as a non-limiting example ofthe instant invention.

Yeast strains AB122, JSC310, and AD2 (Chiron Corporation) weretransformed with the appropriate MEFA expression plasmid (such aspMEFA-6) using a lithium acetate protocol. Ura⁻ transformants werestreaked for single colonies and patched onto Leu⁻/8% glucose plates toincrease plasmid copy number. Leu⁻ starter cultures were grown for 24hours at 30° C. and then diluted 1:20 in YEPD (yeast extractbactopeptone glucose) media. The cells were grown for 48 hours at 30° C.and harvested. To test for expression of the MEFA-6 recombinant antigen,an aliquot of the cells (0.5 OD unit equivalent) was boiled in SDS(sodium dodecylsulfate) gel electrophoresis sample buffer (e.g. Lammlibuffer) containing 50 mM DTT and the protein components of the cellmixture were separated by gel electrophoresis on an Tris-glycinepolyacrylamide gel. MEFA-6 was highly enriched in the insoluble pelletfraction.

Purification of a MEFA Protein in Yeast

The following procedure describes the purification of a specific MEFA,MEFA-6. The techniques and conditions are not intended to limit theinvention, as one of ordinary skill in the art may find it necessary toadjust conditions for the purification of another MEFA of the invention.Unless otherwise indicated, purification of a MEFA is conducted atapproximately 0° C.

MEFA-6 was expressed in S. cerevisiae and cells were harvested asdescribed above. The cells were suspended in lysis buffer (50 mM Tris,0.15 M NaCl, 1 mM EDTA, 1 mM PMSF, pH 8.0) and lysed in a Dyno-Mill (WabWilly A. Bachofon, Basel, Switzerland) or equivalent apparatus usingglass beads. The lysate was centrifuged at low speed conditions (3,000to 5,000 rpm, 15 min) and the pellet containing the insoluble proteinfraction was washed with increasing concentrations of urea (1 M, 2 M, 3M) in lysis buffer. Protein was solubilized from the centrifugationpellet with 0.1 N NaOH, 4 M urea in lysis buffer. Cell debris wasremoved by low speed centrifugation at 3,000 to 5,000 rpm, 15 min. Thesupernatant was adjusted to pH 8.0 with 6 N HCl to precipitate proteinsinsoluble under these conditions.

The precipitate was removed by centrifugation and the supernatant wasadjusted to 2.3% SDS, 50 mM DTT, pH 8.0 and boiled for 3 min. Proteinsin the mixture were fractionated by gel filtration on a PharmaciaSephacryl S-400 in phosphate buffered saline containing 0.1% SDS, 1 mMEDTA and adjusted to pH 7.4. Column eluate fractions containing MEFA-6were collected, pooled, and concentrated on an Amicon YM-30 membrane.Gel filtration was repeated on the pooled fractions using the samecolumn and conditions.

Evaluation of the Antigenicity of a MEFA

In order to evaluate the antigenicity of a chimeric antigen of theinvention, the epitopes of MEFA-3 were exposed to polyclonal ormonoclonal antibodies raised to specific individual epitopes. Purifiedrecombinant multiple epitope fusion antigens (MEFA) were diluted tooptimal coating concentration in phosphate-buffered saline (pH 7.4) andcoated on Immulon I plates (Dynatech). Monoclonal antibodies to core,NS3 (c33c), NS4 (c100 and 5-1-1), NS5 and polyclonal antisera anti-E1and E2 from rabbits were prepared by standard techniques (BIOS-Chile,Maraton 1943, Santiago, Chile) and were diluted 200-fold in samplediluent on the plate and incubated for 1 hr at 37° C., and washed withplate wash buffer (PBS, 0.075% Tween-20, pH 7.2). Either goat anti-mouseF(ab′)₂ or affinity purified goat anti-rabbit IgG heavy and light chainspecific antibody conjugated to horseradish peroxidase (diluted 1:5000for anti-mouse conjugate; diluted 1:10,000 for anti-rabbit conjugate)were added to each assay well. The plates were incubated for 1 hr at 37°C. and washed. o-Phenylenediamine dihydrochloride (OPD) and hydrogenperoxide were added for horse radish peroxidase (HRPO) reaction colordevelopment. The optical density readings were determined using a platereader at 492/620 nm.

The results indicated that all of the antigen epitopes within thedesigned MEFA were easily detected by the specific HCV antibodies forall of the MEFAs of the invention. For example, Table 3 provides data onthe immunoreactivity of the individual epitopes, as well as the chimericantigen, MEFA-3, to monoclonal antibodies of HCV-specific epitopes. Asshown in Table 3, core, c33c, c100, 5-1-1, and NS5 epitopes of MEFA-3were immunoreactive with HCV-specific antibodies. Table 4 shows that theepitopes c33c, c22, 5-1-1, c100, NS5, E1, and E2 of MEFA-5 and MEFA-6were immunoreactive with HCV-specific antibodies.

TABLE 3 HCV Specific Epitopes of MEFA-3 Antigen: Evaluation by Anti-HCVMonoclonal Antibodies HCV Mab ID# 3G1-1 4D1-1 22AFG3 20AGF3 5A1/F5Comment Results Mab Specificity anti-core anti-c33c anti-5-1-1 anti-c100anti-ns-5 Recombinant OD OD OD OD OD Test antigens SOD (non- 0.001 0.0010.002 0.002 0.003 No reaction with SOD recombinant) C25 2.755(+)2.813(+) 2.726(+) 0.028(−) 0.023(−) React with epitopes of core, c33c &5-1-1 c22 (core) 2.700(+) 0.043(−) 0.035(−) 0.036(−) 0.038(−) React withepitope of core c33c (NS3) 0.029(−) 2.646(+) 0.018(−) 0.020(−) 0.014(−)React with epitope of c33c c100 (NS4) 0.020(−) 0.022(−) 2.907(+)3.021(+) 0.016(−) React with epitopes of 5- 1-1 and C-terminal epitopeof c100 NS5 0.012(−) 0.029(−) 0.009(−) 0.009(−) 2.513(+) React withepitope of NS5 Test Antigen 3.236(+) 3.236(+) 3.467(+) 0.713(+) 0.024(−)React with epitopes of MEFA-3 core c33c, 5-1-1 and c100

TABLE 4 HCV Epitope Exposure Within MEFA-5 and MEFA-6 MEFA-6 MEFA-5Antigenic to HCV epitope exposure epitope exposure Antibody ID AntibodySpecifity sequence region OD OD Mab 3G1-1 anti-core (c22c) (aa# 10–50)3.018 (R) 2.702 (R) Mab 4D1-1 anti-NS3 (c33c) linear epitope of c33c3.119 (R) 2.952 (R) Mab 6C10/D1 anti-NS4 (c100) (aa# 1901–1940) 3.853(R) 2.998 (R) Mab 22A5/C12 anti-NS4 (5-1-1) (aa# 1689–1735) 3.006 (R)3.192 (R) Mab 3E1/F1 anti-NS5 (aa# 2297–2313) 2.808 (R) 2.863 (R) Mab1E5/F10 anti-NS5 (aa# 2297–2313) 2.892 (R) 2.784 (R) polyclonal R667anti-E1 (aa# 192–380) 4.375 (R) 1.908 (R) polyclonal R669 anti-E2 (aa#404–662) 1.76 (R) 0.963 (R) Cutoff value 0.45 OD 0.45 OD R = Reaction NR= No Reaction

Inhibition Assays: Peptide inhibition assays were performed to testwhether serotype specific epitopes on a MEFA antigen detect HCVtype-specific antibodies in serum. The assay evaluated the degree towhich a MEPA in solution would bind to serum HCV type-specificantibodies, thereby inhibiting the subsequent ELISA reaction in whichthe serotype-specific peptides are the antigenic species on a solidsupport. FIG. 4 is a schematic drawing of a standard ELISA procedure inwhich binding to the solid support-bound antigen is detected by enzymecatalyzed hydrolysis.

Inhibition assays were performed by multi-antigen ELISA. Recombinant HCVantigens were prepared as described in Chien et al. (1992) PNAS89:10011–10015. The c22 (119 amino acids), E1 (130 aa), NS5 (942 aa),and chimeric C25 (858 aa) antigens were expressed as internal antigenswithin the yeast S. cerevisiae as C-terminal fusions with humansuperoxide dismutase (SOD) using methods described previously for thegeneration of the c100-3 (363 aa) antigen (Kuo, G. et al. (1989) Science244:362–364, herein incorporated by reference; and Cousens, L. S. et al.(1987) Gene 61:265–275, herein incorporated by reference). The c33cantigen (363 amino acids) was expressed as an internal SOD fusionpolypeptide in E. coli by methods described for the synthesis of the5-1-1 antigen (Choo, O.-L. et al. (1989) Science 244:359–362, hereinincorporated by reference). The recombinant HCV antigens were purifiedas described in Chien, D. Y. et al. ((1989) PNAS 89:10011–10015, supra),herein incorporated by reference).

Prior to performing the inhibition assays, the patient sample dilutionbreaking points were determined (Table 5). Patient samples were seriallydiluted and tested for reaction to recombinant c22, c33c, c100 and NS-5antigens immobilized separately onto a solid support (see, for example,Van der Poel, C. L. et al. (1991) Lancet 337:317–319, hereinincorporated by reference). The dilution breaking point was the greatestdilution at which binding was still detectable. For optimal detection insubsequent inhibition assays, the patient samples were less dilute thanthe dilution breaking point dilution, as indicated in Table 6.

TABLE 5 Detection Limit Determination for Patient Samples MEFA-3 AntigenEpitopes Sample Dilution Breaking Points HCV Patient Sample IDRecombinant Antigens PAA LL57366 c22 c33c c100 NS5 1:8 1:128 neat neatPAA LL57454 1:32 1:128 1:8 neat PAA FF25946 1:32 1:256 1:32 NR PAAFF25912 ND ND ND neat NR = no reaction ND = not done

In general, the inhibition assays were performed by the followingprocedure. Recombinant HCV antigens and denatured SOD (control) werediluted to optimal concentration in phosphate-buffered saline (pH 7.4)and coated on Immulon I plates (Dynatech). A 200 μl aliquot of either30% fetal calf serum (FCS) or MEFA-3 peptide (5 or 10 μg per assay asindicated) dissolved in 30% FCS was mixed on the plate with 5 μl ofdiluted serum or plasma specimen. The samples were incubated for 1 hr at37° C. and washed with plate wash buffer. Polyclonal goat anti-human IgG(heavy- and light-chain-specific) antibody conjugated to either ¹²⁵I orhorseradish peroxidase (HRP) was added to each well. The plates wereincubated for 1 hr at 37° C. and then washed. o-Phenylenediaminedihydrochloride and hydrogen peroxide were added for HRP colordevelopment. The results were read using a plate reader at 492 nm/620 nm(ELISA). The ELISA cutoff OD values for antigens from regions SOD, C25,c22, E1, E2, c33c, and NS-5 were 0.40 plus the mean OD of three negativecontrol sera included in each assay. If the control SOD antigen wasreactive, then that sample was considered to be nonreactive orindeterminate. The percentage of binding inhibition was calculated bythe following formula: 100×(A492 nm for patient sample without addedMEFA antigen)−(A492 nm for patient sample with added MEFA antigen)/(A492nm for patient sample without added MEFA antigen). The % inhibition ofbinding to type specific peptides caused by added MEFA-3 indicates thatthe ability of the epitopes within MEFA-3 to bind the anti-HCVantibodies of the patient samples (See Table 6).

TABLE 6 Binding Inhibition by Specific Epitopes of MEFA-3 MEFA-3 PatientSample Control Added ID Dilution OD OD % Inhibition c22 Antigen LL573661:4 1.614 0.163 90% LL57454 1:16 1.370 0.212 84.5% FF25946 1:16 2.0130.205 90% c33c Antigen LL57366 1:64 2.525 0.07 99% LL57454 1:64 1.8390.075 96% FF25946 1:128 0.842 0.061 93% c100 Antigen LL57454 1:4 1.6660.484 71% FF25946 1:16 2.364 0.092 96% NS-5 Antigen LL57454 Neat 2.3191.820 20% FF25912 Neat 1.490 0.873 41%

The ability of MEFA-3 to interact with anti-HCV type 1 and anti-HCV type2 antibodies was demonstrated by inhibition studies using a MEFA ELISAprotocol. Individual synthetic peptides from HCV type 1a, 1b, 2a, and 2b5-1-1 regions were immobilized on separate solid supports. The abilityof the synthetic peptides from the 5-1-1 region to bind the typespecific patient antibodies was determined by competition with addedMEFA-3. The results in Table 7 show that MEFA-3 inhibits binding of HCV1a, 1b, 2a, and 2b to the individual type specific epitopes (amino acids1689–1718 from the 5-1-1 region). The ability of a MEFA to bindantibodies to two different strains of HCV was the same for MEFA-3,MEFA-5, and MEFA-6.

TABLE 7 HCV Type Specificity: MEFA-3 5-1-1 Epitopes Interact WithAntibodies to HCV Types 1 and 2 Control HCV Type Specific Peptides HCV1aHCV1b HCV2a HCV2b (1689–1718) (1689–1718) (1689–1718) (1689–1718)Inhibition, % epitope epitope epitope epitope HCV Type SpecificPeptide + MEFA-3 specific specific specific specific HCV1a HCV1b HCV2aHCV2b ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA Sample OD OD OD ODOD OD OD OD (A) HCV-Type 1 Sample  #4(1:10d) 1.093 0.073 0.002 0.004 0.165  0.136  0.044  0.014 % Inhibition 85%  0%  0%  0% (B) HCV-type 1bsample #358 0.964 1.543 0.424 0.235  0.438  0.261  0.284  0.234 %Inhibition 55% 83% 33%  0% (C) HCV-type 2 sample  #32(1:10d) 0.001 0.0010.839 0.460  0.007  0.018  0.034  0.055 % Inhibition  0%  0% 96% 88%

Example 2 Sensitivity of ELISA Using a MEFA as the Antigen

A comparison of dilution sensitivity was made between MEFA ELISA(MEFA-3) and C25 ELISA. HCV polyprotein C-25 (c33c-c100-3-c22) and assayprocedures were as described by Chien, D. Y. et al. (1992) PNAS USA89:10011–10015, supra) using a coating buffer of 1×phosphate bufferedsaline (PBS), pH 7.0–7.2. Antigens were coated onto the surface ofImmulon I plate microliter wells at 100 ng antigen per well plus 5 μg/mlBSA. Sample size was 5 μl per assay. The goat anti-human IgG (heavy- andlight-chain-specific) antibody conjugated to horse radish peroxidase wasdiluted 1:60,000 for the MEFA-3 assay, and 1:40,000 for the C25 assay.The results in Table 8 show that serum antibodies are detectable usingMEFA-3 ELISA at dilutions at which the C25 ELISA showed no reaction. Thesensitivity of MEFA-3, -5, and -6 CLIA were compared to each other andto C25 ELISA. The results in Table 9 show that MEFA-5 and MEFA-6 CLIAprovided superior sensitivity to MEFA-3 CLIA, while MEFA-3 CLIA was moresensitive than C25 ELISA.

TABLE 8 Dilution Sensitivity: Comparison Study between MEFA ELISA andC-25 ELISA MEFA-3 ELISA C-25 ELISA Immulon I plate Immulon I plate 100ng/well + 5 ug/ml BSA 100 ng/well + 5 ug/ml BSA Conjugate: 1:60000Conjugate: 1:40000 Sample size: 5 ul/assay Sample size: 5 ul/assaySample Panel ID OD OD Sample Dilution LL57454 1:512 0.983 0.734 1:10240.652 NR 1:2048 0.463 NR LL57366 1:512 0.609 0.425(+/−) 1:10240.522(+/−) NR 1:2048 0.203 NR FF25946 1:100 1.818 1.736 1:1000 0.7630.525 1:2000 0.718 NR 1:4000 0.455 NR Seroconversion Panel C Bleed DateC7 (8/29/88) day 1 0.562 NR C8 (9/01/88) day 4 1.035 0.667 C9 (9/28/88)day 32 2.762 2.145 Men of negative 0.124 0.086 sample OD Cutoff OD 0.550.45 (+/−) = OD near cutoff value NR = Non-reactive C-25 ELISA isequivalent to 2G (Second Generation) HCV ELISA

TABLE 9 Dilution Sensitivity of MEFA-3 vs. −5 vs. −6 vs. c25 SENSITIVITYPANEL MEFA-3 CLIA MEFA-5 CLIA MEFA-6 CLIA c25 ELISA Patient SampleS/C.O. S/C.O. S/C.O. S/C.O. FF25946 1:16 1.71 2.72 2.67 1.32 1:32 1.642.59 2.48 1.35 1:64 1.50 1.89 2.11 1.20 1:128 1.34 1.92 1.68 0.92 1:2561.11 1.48 1.68 0.91 1:512 0.84 1.14 1.28 0.69 1:1024 0.58 0.82 1.11 0.63LL57385 1:16 1.73 2.74 2.68 1.49 1:32 1.56 2.41 2.18 1.04 1:64 1.20 1.761.79 1.00 1:128 0.87 1.10 1.03 0.61 1:256 0.76 0.93 0.90 0.57 1:512 0.510.68 0.64 0.48 1:1024 0.38 0.47 0.45 0.39 1:2048 0.23 0.33 0.29 0.20FF25879 1:16 1.70 2.79 2.54 1.46 1:32 1.66 2.73 2.38 1.03 1:64 1.30 1.821.88 0.86 1:128 1.21 1.35 1.17 0.73 1:256 0.96 1.20 1.14 0.66 1:512 0.600.88 0.73 0.52 1:1024 0.48 0.76 0.36 0.50 1:2048 0.42 0.65 0.44 0.40LL57366 1:16 1.67 2.71 2.59 1.59 1:32 1:32 2.30 1.92 1.15 1:64 1.11 1.651.57 0.96 1:128 1.19 1.35 1.09 0.77 1:256 0.84 1.02 1.11 0.63 1:512 0.550.83 0.88 0.50 1:1024 0.55 0.60 0.54 0.47 1:2048 0.38 0.49 0.58 0.37LL57454 1:16 1.87 3.10 2.59 1.80 1:32 1.57 2.82 2.16 1.33 1:64 1.30 2.171.38 1.14 1:128 1.11 1.66 1.38 0.79 1:256 0.63 1.07 1.04 0.60 1:512 0.510.76 0.74 0.43 1:1024 0.41 0.52 0.54 0.34 1:2048 0.22 0.45 056 0.30 S/CO= sensitivity (OD)/cutoff (OD)

A seroconversion sensitivity assay measures the sensitivity of themethod to detecting pathogen-specific antibodies as the titers increasein response to infection. The sensitivity of MEFA-3 ELISA compared toC25 ELISA for blood samples from a single HCV-infected patient over timeis provided in Table 8. MEFA-3 detected antibodies with greatersensitivity at an earlier time post-infection that the C25 ELISA.

Sensitivity and Convenience of a Chemiluminescence Immunoassay UsingMEFA Relative to an Existing Commercial Assay

MEFA as Tracer.

MEFA-6 recombinant antigen was used to design a manual chemiluminescenceimmunoassay (CLIA) as well as an automated CLIA on the Ciba CorningACS-NG system (F-model).

A CLIA, designated the HCV r-Ag-DMAE CLIA (HCV recombinantantigen-dimethyl acridinium ester chemiluminescence immunoassay) wasdeveloped (FIG. 5). A polypeptide or synthetic peptide antigen waslabeled with DMAE by reaction of amino acid side chains (e.g. lysine eside chain or cysteine thiol) with a reactive moiety covalently linkedto DMAE (see WO 9527702, published Oct. 19, 1995, Ciba CorningDiagnostics Corp., herein incorporated by reference). The HCV MEFAsdescribed herein were labeled by reaction with the amino groups oflysine side chains with NSP-DMAE-NHS(2′,6′-Dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl10-(3′-Sulfopropyl)-acridinium-9-carboxylate) obtained from CibaCorning. Thiols of amino acid side chains can be labeled usingDMAE-ED-MCC or NSP-DMAE-PEG-BrAc (Ciba Corning). Labeling procedureswere generally as described in WO 9527702 (supra) with variations inconditions as necessary for each antigen to provide optimal detectionand antigenicity. It is understood that other detectable markers areuseful in the invention, such as fluorescent compounds, rhodaminecompounds, antibodies, antigens, enzymes, and the like. Labeling withany marker is carried out under conditions for obtaining optimaldetection and antigenicity of the of MEFA or other epitope.

Where DMAE is the detectable marker in an assay, the resultant HCVr-Ag-DMAE conjugate is the tracer, with DMAE detectable by lightemission when reacted with NaOH/H₂O₂. When a particular MEFA, such asMEFA-6, was used in the assay, it was designated the MEFA-6-DMAE CLIA.

Manual assay. A manual HCV r-Ag-DMAE CLIA protocol used for the studiesdisclosed herein is first described. A Magic Lite Analyzer System II(MLA II) was used for the manual assay. Parameters such as volume,concentration, time, and temperature are provided for guidance.Variation of these parameters to obtain antibody detection is within thescope of the invention. A 2–10 μl aliquot of test sample was added tocorresponding tubes. The test sample was preferably a biological fluid(plasma or serum, for example) containing anti-HCV antibodies. To eachtube was added 50 μl of water followed by 100 μl biotinylatedrecombinant antigens, synthetic peptides, or directly conjugate DMAE tothe polypeptides (MEFA-6-DMAE, c33c-DMAE, c200-DMAE, and c22-DMAE, forexample). The antigens were diluted in ligand reagent (LR) diluent toconcentrations from approximately 0.1 μg/assay to 1 μg/assay.Preferably, an amount of ligand reagent was added to each sample suchthat approximately 25×10⁶ light unit equivalents (relative light units,RLU) were present per assay. This approximate amount of light unitequivalents was preferred for the addition of a single ligand, or formultiple ligands. LR diluent contained Tris buffer, pH 8.0, 150 mM NaCl,1.0% BSA, 0.1% Tween-20, 0.09% NaN₃, 1 mM EDTA. A 150 μl aliquot of PMP(paramagnetic particles) attached to anti-human IgG Fc was added to eachtube for a final concentration of 60 μg/assay. Preferably, theparamagnetic particles were less than approximately 10 μm in diameter.The anti-IgGFc-PMP particles were diluted in a diluent containing Trisbuffer, pH 8.0, 150 mM NaCl, 2.75% BSA, 0.1% casein, 0.1% Tween-20, 0.1%yeast extract, 0.25% E. coli extract, 0.005% SOD, 0.09% NaN₃, 1 mM EDTA.To ensure complete mixing, the tubes were shaken on a Vortex mixer 6times at 5–10 seconds each time. The sample tubes were incubated at 37°C. for 18 minutes. The sample tubes were placed on a magnet for 3minutes, for sufficient time to sediment the PMP particles. The sampleswere decanted using a magnet to retain the PMP particles. The PMPparticles were washed twice with vortexing in 1 ml of PBS. The washsolution was PBS, 0.1% Tween-20, 0.09% NaN₃, 1 mM EDTA. The steps ofmixing, incubating, sedimenting and decanting may be repeated at leastone time. To each tube 100 μl of water was added to resuspend the PMPparticles. The tubes were then placed in an MLA-II instrument and lightemission was measured for 2 seconds.

The manual MEFA-6-DMAE CLIA method provided enhanced detectionsensitivity relative to the MEFA-6 ELISA. Following the study of eightdilution sensitivity panels, it was found that the MEFA-6-DMAE CLIAdemonstrated a better dilution sensitivity than ELISA in six out ofeight panels.

Importantly, the MEFA-6-DMAE CLIA method detected the presence of HCVantibodies in all samples from chronically infected HCV patients tested.For example, of 29 chronic hepatitis C infected individuals, 26 testedpositive using a C25 ELISA, while all 29 tested positive using theMEFA-6-DMAE CLIA of the invention. In addition, no false positiveresults were found during the testing of 200 random samples byMEFA-6-DMAE CLIA. Other advantages of the CLIA method are inter-assayand intra-assay precision with covariences of less than 10%. Inaddition, the CLIA had a wider response range and improved linearityrelative to ELISA.

Automated Assay. An automated MEFA-DMAE assay having the followingprotocol was also used. An F model automated analyzer was used for theassay. A 10 μl sample (such as a biological fluid containing humananti-HCV antibodies) was added to each sample tube. The automatedsampler then simultaneously dispensed into each sample tube thefollowing: 100 μl of HCV r-Ag-DMAE conjugate (having a total ofapproximately 25×10⁶ light unit equivalents per test) plus 150 μlanti-human IgGFc attached to paramagnetic particles (60 μg IgGFc perassay) plus a 40 μl water backing. The ligand diluent and the IgG-PMPdiluent were as described above for the manual assay. No mixing byvortex was required. The samples were heated to 37° C. for 18 min on aheating block. The anti-human IgG FC PMP particles which bound to theHCV antibodies present in the serum sample were washed three times withresuspension in a wash buffer of PBS containing 0.1% Tween 20, 0.09%NaN₃, 1 mM EDTA. A magnet was used to retain the PMP particles while thesample supernatants were aspirated. The particles were resuspended in500 μl wash buffer. Using the automated method, it was not necessary torepeat the mixing, incubating, sedimenting, and decanting steps therebymaking the HCV r-Ag-DMAE CLIA assay both efficient (20 minutes versus 40minutes), sensitive, and accurate relative to existing commercialassays.

The MEFA-6-DMAE CLIA and the MEFA-6-DMAE +c33c-DMAE CLIA had better orequivalent sensitivities and specificities when compared to themultiantigen HCV 2.0G ELISA tests (Chiron Corp., Emeryville, Calif.),which contain the separate recombinant peptides c100-3, c22-3, and c200(c33c linked to c100-3) (see FIG. 7). Further, the assay method of theinvention is easy to perform because it is a one-step simultaneous assayon a single instrument using one convenient, recombinant captureantigen. According to further embodiments of the invention theadditional epitope may a different epitope of the MEFA, such asconformational epitopes CHO E1 or CHO E2 (HCV epitopes E1 or E2expressed from chinese hamster ovary cells) and labeled with adetectable marker as described for additional epitope c33c in the aboveexample. Such conformational epitopes from HCV and immunoassaysinvolving them are described in WO 96/04301, WO 94/01778, WO 95/33053,WO 92/08734, supra.

Seroconversion Sensitivity

The seroconversion sensitivity of the MEFA-6 chimeric antigen was alsodetermined by CLIA (DMAE as detectable marker) and compared tocommercial ELISA methods. In addition to using the MEFA-6-DMAE alone asan antigen, a mixture of MEFA-6-DMAE +c33c-DMAE was tested forseroconversion sensitivity as another embodiment of the invention. Bloodsamples were obtained from a chronically infected HCV patient over time,tested by CLIA using the procedure described above, and compared withthe performance of Ortho 3.0 EIA (ELISA) (Table 10, only) and Abbott 2.0ELISA (see FIG. 8 and Table 10). Sensitivity was reported as the opticaldensity of the assay sample divided by the assay detection cut off inoptical density units (S/CO).

The detection of HCV antibody in these samples was also performed by acommercial strip immunoblot assay (RIBA® 3.0 Chiron Corporation), whichassay is used clinically as a confirmatory test for HCV antibodydetection. According to the RIBA® method, recombinant HCV antigens areseparated by gel electrophoresis and contacted with patient serum.Reactivity with the separated antigens is performed by immunoblot assayusing secondary labeled antibodies (Eheling, F. et al. (1991) Lancet337:912–913).

The results of the comparison in FIG. 8 and Table 10 indicate that theMEFA-6-DMAE+c33c-DMAE assay was able to detect HCV antibodies withgreater sensitivity at an earlier bleed date. The MEFA-6-DMAE andMEFA-6-DMAE+c33c-DMAE assays were more sensitive at earlier bleed timesthan either the commercial assays or the confirmatory RIBA® test.

The MEFA CLIA method of the invention was compared to ELISAs fromcommercial sources to confirm that the MEFA CLIA reliably detects truepositive and true negative samples. The results in FIG. 9 show that theHCV antibody detection using MEFA CLIAs of the invention is consistentlycorrelated with the antibody detection of the HCV Second GenerationELISA used commercially (Abbott Laboratories). In the cases where asample was assayed as positive for HCV antibodies by the commercialassay and negative by the MEFA CLIA, the sample was found to be negative(non-reactive) by the confirmatory RIBA^(3.0) test, further supportingthe accuracy of the MEFA CLIA of the invention.

TABLE 10 Seroconversion Sensitivity Patient MEFA 6 + Bleed MEFA-6 c33cOrtho 3.0 Abbott 2.0 Day CLIA CLIA ELISA ELISA RIBA* 3.0 1 0.63 0.930.02 0.2 0 (Nonreactive) 2 0.63 0.94 0.02 0.2 0 (Nonreactive) 7 0.631.17 1.45 0.4 I (Intermediate) 9 0.74 1.27 2.74 0.8 I (Intermediate) 141.99 3.54 4.11 3.9 I (Intermediate) 16 3.64 6.38 4.11 5 I (Intermediate)20 6.84 10.9 4.11 5.3 4 (Reactive) Seroconversion panel ID: BostonBiomedical, Inc. anti-HCV Serconversion panel (PHV902)

The accuracy of detection of HCV antibodies was further demonstratedusing MEFA-6-DMAE CLIA (see FIG. 10). Two hundred random negativesamples from blood donation centers and 42 known HCV positive sampleswere tested using the MEPA-DMAE CLIA protocol described above. As FIG.10 indicates, no false positives were found when testing the negativesamples, and no negative results were obtained when testing the knownpositive samples.

Biotinylated MEFA.

A chemiluminescence immunoassay (CLIA) was developed in which a MEFA wasattached to biotin as a detectable marker and indirectly attached toDMAE via a biotin-strepavidin-DMAE link. According to this method,anti-human IgGFc-PMP particles as described above were contacted with abiological fluid containing human anti-HCV antibodies. The humanantibodies were bound to the anti-human IgGFc-PMP particles and theMEFA-biotin was bound to the human anti-HCV antibodies. Strepavidin-DMAEconjugate was then bound to the MEFA-biotin. Approximately 25×10⁶ lightunit equivalents of the strepavidin-DMAE were added to each test sample.Unbound material was washed from the sample and the light emitted by thereaction of the PMP particle bound DMAE with NaOH/H₂O₂ was measured for2 seconds.

This MEFA CLIA method differs from the MEFA-DMAE CLIA also describedherein in that the latter has the DMAE tracer molecule attached directlyto the MEFA, whereas the biotinylated MEFA CLIA involves an additionalbiotin/strepavidin link to bind the DMAE tracer molecule to theanti-HCV/MEFA complex. A diagrammatic representation of the assayprocedure is provided in FIG. 6.

The CLIA in which a MEFA is attached to biotin can be automated asdescribed for the MEFA-DMAE CLIA described above. Under thesecircumstances, strepavidin-DMAE would be added to the sample for bindingand detection. Approximately 25×10⁶ light unit equivalents of thestrepavidin-DMAE conjugate are preferably added to the test mixture.

The instant invention has been shown and described herein and wasconsidered to be the most practical, and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

1. A method for detecting antibodies to hepatitis C virus (HCV) in a biological sample suspected of containing antibodies, the method comprising the steps of contacting said sample with a multiple copy epitope sequence comprising the general structural formula (I): (A)_(x)−(B)_(y)−(C)_(z)  (I) wherein (I) is a linear amino acid sequence; (B) is an amino acid sequence containing at least five and not more than 1,000 amino acids which amino acids correspond to a naturally occurring antigenic determinant of a hepatitis C virus (HCV) polyprotein; (A) and (C) are each amino acid sequences different from (B) and different from each other and are each independently an amino acid sequence containing at least five and not more than 1,000 amino acids which amino acids represent an antigenic determinant that is not adjacent to (B) in naturally-occurring strains of HCV; x is an integer of 2 or more and at least two (A)s are the same antigenic determinant from the same HCV strain; y is an integer of 2 or more and at least two (B)s are the same or an equivalent antigenic determinant from different HCV strains; and wherein (A), (B) and (C) are in any linear order; under conditions that permit antibody-antigen reaction; and detecting the presence of immune complexes of said antibodies and said antigens.
 2. The method of claim 1, wherein (A), (B), and (C) are epitopes from a single organism.
 3. The method of claim 1, wherein (A), (B), and (C) are epitopes from 2 organisms.
 4. The method of claim 1, wherein y is 2 or more and at least one (B) is an equivalent antigenic determinant from a different strain of said organism.
 5. The method of claim 1, wherein copies of any one or more of(A), (B), and (C) are identical amino acid sequences when any one or more of x, y and z are greater than
 1. 6. The method of claim 1, wherein (A), (B), and (C) are in a linear order that is different from the linear order of the naturally occurring antigenic determinants.
 7. The method of claim 1, wherein y is 3 and wherein one (B) is an equivalent antigenic determinant from a hepatitis C strain selected from the group consisting of HCV-1, HCV-2, and HCV-3.
 8. The method of claim 1, wherein (A), (B), and (C) are epitopes of a hepatitis C virus, wherein y is 3 and wherein each (B) is an equivalent antigenic determinant from HCV-1, HCV-2, and HCV-3.
 9. The method of claim 8, wherein x is 2 and each (A) is an epitope from the core region of the HCV polyprotein.
 10. The method of claim 8, wherein each (B) is an epitope from the 5-1-1 region of the HCV polyprotein and x is 2, and each (A) is an epitope from the core region of the HCV polyprotein.
 11. The method of claim 8, wherein (B) is an epitope from the 5-1-1 region of the HCV polyprotein and z is 2, and each (C) is an epitope from the core region of the HCV polyprotein.
 12. The method of claim 1, wherein (B) is from the NS3 region of an HCV polyprotein.
 13. The method of claim 1, wherein z is not zero.
 14. The method of claim 1, wherein any of (A), (B), and (C) are separated by one or more amino acids.
 15. The method of claim 14, wherein (A), (B), and (C) are separated by one or more amino acid sequences containing at least five and not more than 1,000 amino acids, which amino acids correspond to an antigenic determinant, and wherein (A), (B), and (C) are not positioned relative to each other in this manner in nature.
 16. The method of claim 1, wherein (A), (B), and (C), are epitopes from regions of the HCV polyprotein, wherein said regions are selected from the group consisting of NS3, NS4, NS5, c100, C25, core, E1, E2, c33c, c100-3, and c22.
 17. The method of claim 1, wherein the multiple epitope polypeptide comprises the formula of the MEFA-3 antigen as depicted in FIG.
 1. 18. The method of claim 1, wherein the multiple epitope polypeptide comprises the formula of the MEFA-5 antigen as depicted in FIG.
 1. 19. The method of claim 1, wherein the multiple epitope polypeptide comprises the formula of the MEFA-6 antigen as depicted in FIG.
 1. 20. The method of claim 1, further comprising the step of coating the multiple epitope polypeptide on a surface of a solid matrix.
 21. The method of claim 20, wherein the solid matrix is selected from the group consisting of the surface of a microtiter plate well, a bead and a dipstick.
 22. The method of claim 1, further comprising the step of attaching a detectable marker to the multiple epitope polypeptide.
 23. The method of claim 22, wherein the detectable marker is selected from the group consisting of a chromophore, an antibody, an antigen, an enzyme, an enzyme reactive compound whose cleavage produce is detectable, biotin, streptavidin, a fluorescent compound, a chemiluminescent compound and combinations thereof.
 24. The method of claim 1, wherein said antibody-antigen complexes are detected by incubating the complexes with a labeled anti-human immunoglobulin antibody.
 25. The method of claim 24, wherein said anti-human immunoglobulin is enzyme labeled.
 26. The method of claim 1, wherein said biological sample is selected from human blood, serum or plasma.
 27. The method of claim wherein 1, said multiple epitope polypeptide is prepared by chemical synthesis.
 28. The method of claim 1, wherein said multiple epitope polypeptide is prepared by recombinant DNA expression. 